Methods of diagnosing multidrug resistant tuberculosis

ABSTRACT

The invention relates to the discovery that a putative gene of  Mycobacterium tuberculosis  with no previously identified function is responsible for the ability of the bacterium to activate thioamide drugs. Since  M. tuberculosis  has a low rate of synonymous mutations, all mutations in this gene, identified as Rv3854 c  and now termed “EtaA,” are expected to inhibit the ability of a bacterium with the mutation to activate a thioamide or thiocarbonyl drug. Thus, detecting a bacterium with a mutation in this gene indicates that the bacterium is resistant to treatment with thioamides.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/888,320, filed Jun. 22, 2001, which claims priority to U.S.Provisional Application No. 60/214,187, filed Jun. 26, 2000. Thecontents of both of these applications are incorporated by reference forall purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

The World Health Organization (“WHO”) estimates that as much asone-third of the world's population is infected with tuberculosis. In1998, the latest year for which estimates are available, Mycobacteriumtuberculosis (“MTb”) infected 7.25 million people and resulted in 2.9million fatalities (Farmer, P. et al., Int J Tuberc Lung Dis 2:869(1998)). Underlying these statistics is an emerging epidemic of multipledrug-resistant (“MDR”) tuberculosis that severely undermines controlefforts and is transmitted indiscriminately across national borders(Viskum, K. et al., Int J Tuberc Lung Dis 1:299 (1997); Bass, J. B. etal., Am J Respir Crit Care Med 149:1359 (1994)). Resistance to any ofthe front-line drugs generally bodes poorly for the patient, who then iscommitted to a regimen of less active “second-line” therapies. Wheremultidrug resistance is suspected, the WHO recommends that three or moredrugs be administered at the same time, to decrease the chance that theorganism will be able to develop resistance to all of the agents.

One of the most efficacious of the second-line drugs is the thioamideethionamide (ETA) (Farmer, P. et al., supra). Like the front-line drug,isoniazid (INH), ETA is specific for mycobacteria and is thought toexert a toxic effect on mycolic acid constituents of the cell wall ofthe bacillus (Rist, N. Adv Tuberc Res 10:69 (1960); Banerjee, A. et al.,Science 263:227 (1994)). Current tuberculosis therapies include a largenumber of “prodrugs” that must be metabolically activated to manifesttheir toxicity upon specific cellular targets (Barry, C. B., III et al.,Biochem Pharm 59:221 (2000)). The best characterized example of this isthe activation of INH by the catalase-peroxidase KatG, generating areactive form that then inactivates enzymes involved in mycolic acidbiosynthesis (Slayden, R. A. et al., Microbes and Infection (2000) (inpress); Heym, B. et al., Tubercle Lung Dis 79:191 (1999)). The majorityof clinically observed INH resistance is associated with the loss ofthis activating ability by the bacillus (Musser, J. M., Clin MicrobiolRev 8:496 (1995)), but such strains typically retain their sensitivitytoward ETA, suggesting that ETA activation requires a different enzymethan KatG (Rist, N., Adv. Tub. Res. 10, 69 (1960)).

In a striking achievement of molecular biology and genetics, the entiregenome of a paradigm M. tuberculosis strain, H37Rv (EMBL/GenBank/DDBJentry AL123456), was sequenced and published in 1998. (Cole, S. et al.,Nature 393,537 (1998)). The genome was found to comprise 4,411,531 basepairs, comprising 3,974 putative genes, of which 3,924 were predicted toencode proteins. Each of the putative genes was accorded a number basedon its position in the genome relative to a selected start site. Thefunction of many of the putative genes, however, could not be determinedwhen the genome was sequenced and published, and their function remainsunknown today.

SUMMARY OF THE INVENTION

The present invention provides methods of determining the ability of aMycobacterium tuberculosis bacterium to oxidize a thioamide orthiocarbonyl, and thereby of determining the resistance of the bacteriumto a thioamide or thiocarbonyl drug or prodrug. The methods include, forexample, detecting a mutation in the EtaA gene in the bacterium, which amutation is indicative of decreased ability to oxidize a thioamide orthiocarbonyl. The wild-type sequence of the EtaA gene is set forth inSEQ ID NO:1. Such mutations can include frameshift, missense, andnonsense mutations, as well as single nucleotide polymorphisms (SNPs)which cause amino acid substitutions in the normal sequence encoded bythe gene. In particular, the frameshift mutations can include, forexample, a deletion at position 65 of the EtaA gene sequence, anaddition at position 557, or an addition at position 811. SNPs canresult in, for example, any of the following amino acid substitutions:G43C, P51L, D58A, Y84D, T342K, and A381P.

The invention further provides methods of detecting such mutations.These methods include, for example, amplifying the EtaA gene, or aportion thereof containing the mutation, with a set of primers toprovide an amplified product, sequencing the amplified product to obtaina sequence, and comparing the sequence of the amplified product with aknown sequence of a wild-type EtaA gene, wherein a difference betweenthe sequence of the amplified product and the sequence of the wild-typeEtaA gene indicates the presence of a mutation. The amplification can beby any of a variety of techniques, such as PCR. For example, the EtaAgene or a portion thereof can be amplified, the amplified product can besubjected to digestion by restriction enzymes, the resulting restrictionproducts can be separated to form a pattern of restriction fragmentlengths, and the pattern of restriction fragment lengths compared to apattern of restriction fragment lengths formed by subjecting thewild-type EtaA gene (or portion thereof corresponding to the portion ofthe EtaA gene amplified from the organism being screened) to the samerestriction enzymes. The amplification can be by PCR.

In preferred embodiments, the primers for amplifying the gene areselected from the group consisting of 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3); 5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4), 5′ ATCATCCATCCGCAGCAC 3′ (SEQ IDNO:5); 5′ AAGCTGCAGGTTCAACC 3′ (SEQ ID NO:6); 5′ GCATCGTGACGTGCTTG 3′(SEQ ID NO:7); 5′ AAGCTGCAG GTTCAACC 3′ (SEQ ID NO:8); 5′TGAACTCAGGTCGCGAAC 3′ (SEQ ID NO:9); 5′ AACATCGTCGTGATCGG 3′ (SEQ IDNO:10); 5′ ATTTGTTCCGTTATCCC 3′ (SEQ ID NO: 11); 5′ AACCTAGCGTGTACATG 3′(SEQ ID NO: 12); 5′ TCTATTTCCCATCCAAG 3 (SEQ ID NO:13); and 5′GCCATGTCGGCTTGATTG 3′ (SEQ ID NO:14). In particularly preferredembodiments, the primers are the sequences of SEQ ID NO:3 and SEQ IDNO:4. The separation of the restriction length fragments can be by gelelectrophoresis. An EtaA gene with a known mutation, such as theparticular mutated EtaA genes described above, can also be amplified andsubjected to restriction enzymes, and the resulting patterns compared tothat of a EtaA gene obtained from a biological sample (for example, froma patient) to determine whether the EtaA gene from the biological samplehas the same mutation as that of the EtaA gene with the known mutation.

The mutations can also be detected by hybridization techniques.Conveniently, the sample nucleic acid is hybridized to a nucleic acid ofknown sequence, such as the wild-type EtaA gene or a portion thereof, orto a portion of the gene containing the mutation, under conditionssufficiently stringent that, if the reference nucleic acid is thewild-type sequence, failure of the sample to hybridize to the referencenucleic acid will indicate that it contains a mutation whereashybridization will indicate it comprises the wild-type sequence. Theconverse will be true if the reference nucleic acid comprises amutation. Either the sample nucleic acid or the reference nucleic acidcan be immobilized on a solid support.

The mutations can further be detected by detecting mutations in the geneproduct. This can be accomplished, for example, by specifically bindingany of a number of antibodies, such as a single chain Fv portion of anantibody or an antibody fragment which retains antibody recognition, toa gene product with a mutation, wherein such binding is indicative of amutation indicating that the organism containing the mutation hasdecreased ability to oxidize a thioamide or thiocarbonyl drug or prodrugcompared to an organism bearing a wild-type EtaA gene. Conveniently, thedetection of specific binding of the antibody and the gene product canbe measured in an ELISA. Mutations can also be detected by massspectrometry. In another embodiment, the mutation is detected byculturing the organism in the presence of ethionamide and testing forthe presence or absence of (2-ethyl-pyridin-4-yl)methanol, wherein theabsence of (2-ethyl-pyridin-4-yl)methanol indicates that the bacteriumhas a mutation which is indicative of decreased ability to oxidize athioamide. Conveniently, the ethionamide may be radiolabeled.

The invention further provides methods for screening an individual withtuberculosis for the presence of a M. tuberculosis bacterium resistantto treatment with a thioamide or a thiocarbonyl drug, comprisingobtaining a biological sample containing the bacterium and detecting amutation in an EtaA gene in the bacterium, wherein detecting thepresence of a mutation is indicative the bacterium is resistant totreatment by a thioamide or a thiocarbonyl drug or prodrug. The methodcan include detecting the mutation by amplification of the EtaA genewith a set of primers to obtain a sequence, sequencing the amplifiedproduct, and comparing the sequence to that of the wild-type EtaA gene,SEQ ID NO:1, wherein a difference between the sequence of the amplifiedproduct and of the sequence of the wild-type gene indicates the presenceof a mutation.

The invention further provides kits for determining the ability of an M.tuberculosis organism to oxidize a thioamide or thiocarbonyl. Such kitsinclude a container and appropriate primers for amplifying an EtaA geneor a portion thereof, and may further comprise one or more restrictionenzymes. In preferred embodiments, the primers for amplifying the geneare selected from the group consisting of 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3); 5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4), 5′ ATCATCCATCCGCAGCAC 3′ (SEQ IDNO:5); 5′ AAGCTGCAGGTTCAACC 3′ (SEQ ID NO:6); 5′ GCATCGTGACGTGCTTG 3′(SEQ ID NO:7); 5′ AAGCTGCAG GTTCAACC 3′ (SEQ ID NO:8); 5′TGAACTCAGGTCGCGAAC 3′ (SEQ ID NO:9); 5′ AACATCGTCGTGATCGG 3′ (SEQ IDNO:10); 5′ ATTTGTTCCGTTATCCC 3′ (SEQ ID NO:11); 5′ AACCTAGCGTGTACATG 3′(SEQ ID NO:12); 5′ TCTATTTCCCATCCAAG 3 (SEQ ID NO:13); and 5′GCCATGTCGGCTTGATTG 3′ (SEQ ID NO:14). In particularly preferredembodiments, the primers are the sequences of SEQ ID NO:3 and SEQ IDNO:4. An EtaA gene with a known mutation can also be included as apositive control.

In other embodiments, the kits may provide materials for performingELISA or immunoassays to detect organisms with decreased ability tooxidize thioamides, or to detect products of thioamide metabolism. Thekits may also contain radiolabeled ethionamide to permit detection oflabeled metabolic products in the presence of an organism which canmetabolize the drug. Moreover, the kits may contain materials forperforming thin-layer chromatography, and may contain(2-ethyl-pyridin-4-yl)methanol for use as a positive control.Alternatively, or in addition, the kits may include an antibody thatbinds to a product of the EtaA gene or to(2-ethyl-pyridin-4-yl)methanol. The kits may also contain instructionsfor detecting mutations in the EtaA gene, such as the specific mutationsidentified above. Detection of such mutations indicates that theorganism has decreased ability to oxidize a thioamide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. In vivo production of (2-ethyl-pyridin-4-yl)methanol (5) fromETA by whole cells of MTb.

FIG. 1A. Metabolism of radiolabeled ETA by MTb. Lanes a-h correspond tosequential supernatant samples taken at times: 0.2, 0.25, 0.75, 1.5,5.0, 8.5, and 25 hours, respectively. Lane i represents mediaautooxidation following 25 hr of incubation without bacterial cells.These metabolites correspond to ETA S-oxide (2), ETA nitrile (3) and ETAamide (4)

FIG. 1B. Cell associated radioactivity counts graphed against time.“DPM,” disintergrations per minute.

FIG. 1C. Left graph. The unknown major metabolite (5) was confirmed as(2-ethyl-pyridin-4-yl)methanol by co-chromatography with a syntheticcharacterized alcohol standard. Right hand graphs. Upper panel: HPLCcontinuous radiodetector spectrum corresponds to FIG. 1A. lane i, mediacontrol. Lower panel: HPLC continuous radiodetector spectrum correspondsto FIG. 1A, lane d, time point 1.5 hr, where the UV254 trace of(2-ethyl-pyridin-4-yl)methanol is superimposed

FIG. 2. EtaA and EtaR control ETA susceptibility and metabolism.Photographs of MSm pMH29 mycobacteria clones grown on 7H11 platescontaining the indicated concentration of drugs. “Control” indicates nodrug added. “INF 12.5” indicates isoniazid was present at 12.5 μg/ml.“ETA 2.5, 12.5, and 62.5” indicate that ethionamide was present at theμg/ml indicated. Within each photograph, the vertical columns show MSmclones which were tranformed with EtaA (a); vector control (b); or EtaR(c), respectively, and spotted in 10-fold dilutions (from top tobottom).

FIG. 3.

FIG. 3A. The MSm clones shown in FIG. 2 were analyzed for their abilityto metabolize [1-¹⁴C]ETA. Lanes a-f correspond to samples taken attimes: 0, 30, 90, 180, 330 and 900 minutes, respectively. Metaboliteswere identified as in FIG. 1.

FIG. 3B. Cell-associated radioactivity was determined as in FIG. 1B.“DPM,” disintegrations per minute. Squares represent MSm overexpressingEtaA, circles represent wild type MSm, triangles represent MSmoverexpressing EtaR.

FIG. 3C. Macromolecule-associated radioactivity. “DPM,” disintegrationsper minute. Columns 1, 2, and 3 show counts for MSm overexpressing EtaA,wild type MSm, and MSm overexpressing EtaR, respectively.

FIG. 4. EtaA and EtaR associated mutations and cross-resistance inpatient isolates from Cape Town, South Africa.

FIG. 4A. Thiacetazone (1) and thiocarlide (2).

FIG. 4B. Map of mutations in EtaA found in patient isolates resistant toETA and thiacetazone. Chromosome coordinates and gene designations arein reference to the sequenced genome of MTb strain H37Rv. The “811” and“65” above the arrow denote nucleotide positions within the genesequence. The notations “+1 nt” and “Δ1 nt” below the arrow denote thatthe patient isolate was found to have a nucleotide added or deleted,respectively, at the position indicated. The other notations below thearrow for the EtaA gene denote, in standard single letter code,substitutions at positions in the amino acid sequence of the geneproduct.

FIG. 4C. Cross-resistance determination of patient isolates and theassociated nucleotide alterations observed. The individual patientisolates are listed vertically in the column entitled “Strain.” Thefinal entry in that column is a mono-resistant strain of MTb (ATCC35830) obtained from the American Type Culture Collection (Manassas,Va.). The next four columns set forth the observed growth of the isolatewhen cultured with the indicated drug. ETA: ethionamide, TA:thiacetazone, TC: thiocarlide, INH: isoniazid. Susceptibility to ETA isreported as follows: S: susceptible (if the culture failed to grow at2.5 μg/ml), L: low-level resistance (if weak growth was observed at 2.5μg/ml), M: moderate resistance (if strong growth was observed at 2.5μg/ml), and H: high-level resistance (if growth was observed at 10μg/ml). Susceptibility to TA/TC/INH is reported as follows: S:susceptible (if the culture failed to grow at 0.5 μg/ml), L: low-levelresistance (if weak growth was observed at 0.5 μg/ml), M: moderateresistance (if weak growth was observed at 2.0 μg/ml), and H: high-levelresistance (if strong growth was observed at concentrations greater than2.0 μg/ml). The column titled “Nucleotide” denotes the position in thenucleotide sequence of the gene at which a mutation, if any, was found.The column titled “Amino-acid” indicates whether the nucleotide mutationdenoted in the column to its left resulted in an amino acid substitutionand, if so, the particular substitution and the position of the affectedamino acid in the normal amino acid sequence of the protein encoded byEtaA.

FIG. 5. The sequence of the EtaA gene. The coding region (SEQ ID NO:1)consists of the 1467 numbered nucleotides. Portions of the untranslated5′ and 3′ regions are shown.

FIG. 6. The amino acid sequence (SEQ ID NO:2) of the protein encoded bythe EtaA gene.

DETAILED DESCRIPTION

Introduction

It has now been discovered that two of the putative genes of M.tuberculosis, Rv3854c and Rv3855, regulate the susceptibility of M.tuberculosis to the major second-line drug, ethionamide (“ETA”), used totreat tuberculosis. Specifically, it has now been discovered that thegene currently known as Rv3854c is a monooxygenase. Further, it has nowbeen discovered that this gene confers upon Mycobacteria the ability toactivate thioamide and thiocarbonyl drugs from their prodrug form totheir active drug form. When the tuberculosis genome was sequenced andanalyzed in 1998, the gene was considered to bear homology to abacterial monooxygenase, but was sufficiently different to be classifiedas a separate, unknown family. Moreover, its substrate was unknown.

It has now further been discovered that the gene currently known asRv3855 is a regulator of expression of the monooxygenase encoded byRv3854c, and can repress its expression. In recognition of the discoveryof the functions of these genes, we have renamed Rv3854c and Rv3855 asEtaA and EtaR, respectively.

It has further been discovered that mutations in the EtaA gene arediagnostic of resistance to ETA. Analysis of patient isolates revealed a100% correlation between mutations in this gene and resistance to ETA.When resistance was selected for, both frameshift mutations, consistingof the deletion or addition of a single nucleotide, and singlenucleotide polymorphisms (“SNPs”) which resulted in the substitution ofone amino acid residue for another, resulted in an ETA-resistantphenotype. It has previously been recognized that M. tuberculosis has anextremely low rate of synonymous mutations; that is, the organism hasfew if any random mutations which do not have a functional effect. E.g.,Sreevatsan, S., et al. Proc Natl Acad Sci USA 94(18):9869-74 (1997).Accordingly, it is expected that any mutation in this gene, whetherframeshift, nonsense, missense, or SNP, will result in an ETA-resistantphenotype. The Examples show that all the mutations studied, includingtwo frameshift mutations and seven SNPs, resulted in increasedresistance to ETA. By contrast, three isolates selected for byresistance to thioacetazone which were not also cross resistant to ETA,and the wild-type strain which showed an ETA-sensitive phenotype, weremutation free in the EtaA/EtaR and intergenic regions. The knowledge ofthe EtaA gene sequence and of the function of the gene permits one ofskill in the art to readily identify any particular mutation of the EtaAgene in an ETA-resistant organism.

It has further been discovered that organisms with mutations in the EtaAgene are resistant not only to ETA, but also to two other thioamidecompounds also used as second-line drugs. Thus, mutations in this genereduce or eliminate the value of at least three of the drugs which havebeen used in combination therapy for MDR tuberculosis. Based on thepresent findings, it can also be predicted that organisms with mutationsin this gene will be resistant to other thioamide- or thiocarbonyl-basedtherapeutic agents.

The extensive cross-resistance among these compounds predicts twooverlapping mechanisms of resistance: (a) target associated, like theresistance between INH and ETA and (b) activation-associated, like theresistance among ETA (a thioamide), thioacetazone (a thioamide), andthiocarlide (a thiocarbonyl). Such considerations complicate appropriatedrug therapy for the treatment of multidrug-resistant tuberculosis andthe discovery of the cross-resistance to these compounds provides animportant tool to help understand the resistance mechanisms operating ina single patient, which may prove vital to determining appropriatetreatment for that patient.

These discoveries permit a much more rapid determination of whether theparticular organism infecting a patient is resistant to thesesecond-line agents. Detection of mutations in the EtaA gene can be usedto diagnose a phenotype resistant to treatment by ETA, and the otherthioamide drugs, thiacetazone and thiocarlide, used as second-lineagents. In addition, the knowledge of the pathway by which ETA ismetabolized permits diagnosis of a drug-resistant phenotype by detectingdifferences in the rate of production of end-products or intermediates.

The diagnosis of a phenotype resistant to thioamide drugs has importantclinical implications. M. tuberculosis tends to develop resistance todrugs when used as single agents (“monotherapy”). Drug-resistanttuberculosis is therefore generally treated with at least two andpreferably three different agents, since it is less likely that theorganism will be able to develop resistance to all three of the agentssimultaneously. ETA is one of the most important drugs recommended bythe World Health Organization for use in the case of multidrug resistant(“MDR”) strains of tuberculosis. If a patient with MDR tuberculosis hasa mutation of EtaA rendering the patient resistant to thioamidetherapies, however, the ETA will have limited or no effect, and it willbe as if the patient has been administered only one or only two agents.The chance that the M. tuberculosis strain present in the patient willdevelop resistance to the other agents is thus higher than expected and,if such resistance develops, no drugs may be left which are capable ofeffectively combating the organism.

Additionally, mutations in the EtaA gene permit rapid identification ofMDR organisms by PCR and other techniques, rather than by having toculture the organisms in the presence of various antibiotics. This isespecially useful because Mycobacteria are such slow growers thatpatients not infrequently die before the Mycobacteria infecting them canbe cultured and their susceptibility determined by conventional means.The rapid identification of organisms permitted by the invention reducesthis problem, and also permits more rapid monitoring of possiblenosocomial spread. Additionally, the prompt confirmation or exclusion ofpossible transmission allows early clinical intervention to prevent orreduce future outbreaks of MDR-tuberculosis.

Definitions and Terms

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; amino acid sequencesare written left to right in amino to carboxy orientation. The headingsprovided herein are not limitations of the various aspects orembodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

Residues mutated from a known sequence are designated by convention bylisting in standard single letter code the residue normally found at adesignated position in the sequence, the position in the sequence of theresidue mutated, and the residue substituted for the original residue.Thus, for example, “G43C” or “G43→C” indicates that a glycine residuenormally found at position 43 of the relevant sequence has been replacedor substituted by a cysteine.

References here to “MTb” refer to Mycobacterium tuberculosis. Thesequence of the entire genome of MTb is set forth in TubercuList, foundon the internet by entering “http://” followed by“genolist.pasteur.fr/TubercuList/”.

References herein to “Rv3854c” and “EtaA” are to a gene found in MTb anddesignated as Accession Number Rv3854c in TubercuList at the web sitenoted above. The EtaA gene is also designated as “EthA”. As used herein,the term “wild-type EtaA gene” and references to the EtaA gene or EthAgene without further elaboration refer to the sequence set forth inTubercuList under Accession Number Rv3854c. The gene has 1467 base pairsand has the following coordinates in the published M. tuberculosisgenome: 4326007 and 4327473. TubercuList lists the gene as encoding a489 amino acid monooxygenase with a molecular weight of 55329.2 and a pIof 8.3315. The published sequences of the EtaA (EthA) gene and of theprotein encoded by the gene are set forth as SEQ ID NO:1 and SEQ IDNO:2, respectively.

The gene described herein as “EtaR” is also designated as “EthR.” It isavailable in TubercuList under accession number Rv3855.

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes where appropriate both polyclonal and monoclonal antibodies.The term also includes genetically engineered forms such as chimericantibodies (e.g., humanized murine antibodies), heteroconjugateantibodies (e.g., bispecific antibodies). The term particularly refersherein to recombinant single chain Fv fragments (scFv), disulfidestabilized (dspv) Fv fragments, or pFv fragments. The term “antibody”also includes antigen binding forms of antibodies, including fragmentswith antigen-binding capability (e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG.See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed., W.H. Freeman & Co.,New York (1998). Which particular sense or senses of the term areintended will be clear in context.

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors, see, e.g., Huse etal., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or byimmunizing an animal with the antigen or with DNA encoding the antigen.

The terms “stringent hybridization conditions” or “stringent conditions”refer to conditions under which a nucleic acid sequence will hybridizeto its complement, but not to other sequences in any significant degree.Stringent conditions in the context of nucleic acid hybridizations aresequence dependent and are different under different environmentalparameters. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, New York, (1993) (the entirety of Tijssenis hereby incorporated by reference). Very stringent conditions areselected to be equal to the T_(M) point for a particular probe. Lessstringent conditions, by contrast, are those in which a nucleic acidsequence will bind to imperfectly matched sequences. Stringency can becontrolled by changing temperature, salt concentration, the presence oforganic compounds, such as formamide or DMSO, or all of these. Theeffects of changing these parameters are well known in the art. Theeffect on T_(m) of changes in the concentration of formamide, forexample, is reduced to the following equation: T_(m)=81.5+16.6 (logNa⁺)+0.41 (% G+C)−(600/oligo length)−0.63(% formamide). Reductions in Tmdue to TMAC and the effects of changing salt concentrations are alsowell known. Changes in the temperature are generally a preferred meansof controlling stringency for convenience, ease of control, andreversibility. Exemplary stringent conditions for detecting singlenucleotide polymorphisms are set forth in numerous references, includingWinichagoon, et al. Prenat Diagn 19:428-35 (1999); Labuda et al., AnalBiochem 275:84-92 (1999); and Bradley et al., Genet Test 2:337-41(1998).

“Solid support” and “support” are used interchangeably and refer to amaterial or group of materials having a rigid or semi-rigid surface orsurfaces. In many embodiments, at least one surface of the solid supportwill be substantially flat, although in some embodiments it may bedesirable to physically separate synthesis regions for differentcompounds with, for example, wells, raised regions, pins, etchedtrenches, or the like. According to other embodiments, the solidsupport(s) will take the form of beads, resins, gels, microspheres, orother geometric configurations.

Detecting Mutations in the EtaA Gene

As noted in the Introduction, MTb is known to have an extremely low rateof “synonymous” mutations; that is, MTh rarely has random mutations thatdo not affect the function of the organism. Thus, any mutation in theEtaA gene is expected to alter the gene sufficiently so that the enzymeencoded by the gene has reduced ability to activate a thioamide prodrug.Thus, any mutation in the EtaA gene carried by an MTb bacillus isindicative of that that organism is resistant to therapy by thioamidedrugs and, in particular, to the thioamide drugs ETA, thiacetazone, andthiocarlide.

There are a number of methods known in the art for detecting mutationsin a given gene. Mutations in the gene can be found directly byamplifying the gene in a MTb of interest and comparing the sequence ofthe organism's gene to that of a reference EtaA gene sequence, such asthe one set forth in TubercuList. Alternatively, one can digest samplesof the EtaA gene of the organism of interest (such as that of a MTbisolated from a patient) and of a known ETA-susceptible MTb organismwith restriction enzymes, separate the resulting fragments byelectrophoretic techniques routine in the art (such as those taught inCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, Greene Publishing Associates, Inc./John Wiley & Sons,Inc., (1994 Supplement) (“Ausubel”)), and compare the pattern of thefragments, with a difference in the pattern of the fragments of thesample compared to that of the EtaA-susceptible organism beingindicative of an impaired ability of the organism to metabolize ETA.This method, known as “restriction fragment length polymorphism,” or“RFLP,” is well known in the art.

The nature of the mutation can be determined by, for example, sequencingthe gene isolated from the individual organism. If the specific mutationfound is not one already identified as resulting in impaired ability ofthe enzyme expressed from the gene, the mutation can be tested by any ofa variety of standard methods to determine the effect of the mutation.For example, the gene can be transformed into a species of Mycobacteriaknown to be somewhat resistant to Eta compared to wild-type (H37Rv), thegene expressed, and the activity of the resulting enzyme compared foractivity against the enzyme expressed by identical cells transformedwith a wild-type EtaA gene. An exemplary assay for transforming cellsand determining the activity of the EtaA enzyme is set forth in theExamples herein.

Another method known in the art is “CFLP,” or “cleavase fragment lengthpolymorphism.” This method involves amplifying the gene of interest,here EtaA, followed by digestion with cleavase I, which cuts the DNA atsites dependent on secondary structure. Results are resolved on agarosegels, forming a “bar code”—like pattern which is indicative of theparticular gene. Different patterns of cleavage digestion products areobtained for wild-type and mutant samples. The technique is sensitiveenough to detect mutations as subtle as point mutations.

Single-stranded conformation polymorphism (“SSCP”)has been used toidentify a number of different drug resistant phenotypes in selectedorganisms.” Line hybridization assays permit identification of mutantforms of genes responsible for resistance after amplification ofrelevant genes by the hybridization patterns of probes to samples.Resistance can be determined, for example, by reverse hybridization lineprobe assay, or “LiPA.” Kits for assays for several genes, such asvarious mutations in the cystic fibrosis gene, are availablecommercially from Innogenetics N.V. (Zwijnaarde, Belgium). For example,in the HLA typing assay, amplified biotinylated DNA is chemicallydenatured, and the single strands are hybridized with specificoligonucleotide probes immobilized as parallel lines on membrane-basedstrips. Then, strepavidin labeled with alkaline phosphatase is added andbound to any biotinylated hybrid previously formed. Incubation with anappropriate substrate results in a precipitate, and the reactivity ofthe probes can be determined.

A further method known in the art is temperature modulation heteroduplexchromatography (“TMHC”). The method involves amplification of the geneof interest, here the EtaA gene, followed by denaturing of the PCRproducts and then slowly cooling, to a predetermined temperature basedon the composition of the sample. While cooling, the PCR productsrenature forming hetero and homoduplexes which are resolved from oneanother using TMHC. Conveniently, the resolution is performed using aWAVE® DNA fragment analysis system (Transgenomic, Inc., San Jose,Calif.).

In another set of embodiments, mutations in the EtaA gene are detectedby hybridizing the gene or portions thereof from a biological sample,such as from an individual, against a reference nucleic acid, such asthe wild-type EtaA gene or one or more EtaA genes with a known mutation(for ease of discussion herein, the reference nucleic acids will betermed “probes” and the sample being screened the “nucleic acid ofinterest”). The hybridizations can be performed while either the probesor the nucleic acids of interest are attached to solid supports, orwhile they are in a fluid environment.

In one set of embodiments, the hybridizations are performed on a solidsupport. For example, the nucleic acids of interest (or “samples”) canbe spotted onto a surface. Conveniently, the spots are placed in anordered pattern, or array, and the placement of where the nucleic acidsare spotted on the array is recorded to facilitate later correlation ofresults. The probes are then hybridized to the array. Conversely, theprobes can be spotted onto the surface to form an array and the sampleshybridized to that array. In another set of embodiments, beads are usedas solid supports. Conveniently, the beads can be magnetic or made ofmaterials responsive to magnetic force, permitting the beads to be movedor separated from other materials by externally applied magnetic fields.

The composition of the solid support can be anything to which nucleicacids can be attached. It is preferred if the attachment is covalent.The material for the support for use in any particular instance shouldbe chosen so as not to interfere with the labeling system to be used forthe probes or the nucleic acids. For example, if the nucleic acids arelabeled with fluorescent labels, the material chosen for the supportshould not be one which fluoresces at wavelengths which would interferewith reading the fluorescence of the labels.

Preferably, the support is of a material to which the samples and probesbind or one which is substantially non-porous to them, so that theoligonucleotides remain accessible (i.e., to the probes or the samples)at the surface of the support. Membranes porous to the nucleic acids maybe used so long as the membrane can bind sufficient amounts of nucleicacid to permit the hybridization procedures to proceed. Suitablematerials should have chemistries compatible with oligonucleotideattachment and hybridization, as well as the intended label, andinclude, but are not limited to, resins, polysaccharides, silica orsilica-based materials, glass and functionalized glass, modifiedsilicon, carbon, metals, nylon, natural and synthetic fibers, such aswool and cotton, and polymers.

In some embodiments, the solid support has reactive groups such ascarboxy- amino- or hydroxy groups to facilitate attachment of theoligonucleotides (that is, the samples or the probes). Plastics may beused if modified to accept attachment of nucleic acids oroligonucleotides (since plastic usually has innate fluorescence, the useof non-fluorescent labels is preferred for use with plastic substrates.If plastic materials are used with fluorescent labels, appropriateadjustments should be made to procedures or equipment, such as the useof color filters, to reduce any interference in detecting results due tothe fluorescence of the substrate). Polymers may include, e.g.,polystyrene, polyethylene glycol tetraphtalate, polyvinyl acetate,polyvinyl chloride, polyvinyl pyrrolidone, buty rubber, andpolycarbonate. The surface can be in the form of a bead. Means ofattaching oligonucleotides to such supports are well known in the art,and are set forth, for example, in U.S. Pat. Nos. 4,973,493 and4,569,774 and PCT International Publications WO 98/26098 and WO97/46313. See also, Pon et al., Biotechniques 6:768-775 (1988); Damba,et al., Nuc. Acids Res. 18:3813-3821 (1990).

Alternatively, the samples can be placed in separate wells or chambersand hybridized in their respective well or chambers. The art hasdeveloped robotic equipment permitting the automated delivery ofreagents to separate reaction chambers, including “chip” andmicrofluidic techniques, which allow the amount of the reagents used perreaction to be sharply reduced. Chip and microfluidic techniques aretaught in, for example, U.S. Pat. No. 5,800,690, Orchid, “Running onParallel Lines” New Scientist, Oct. 25, 1997, McCormick, et al., Anal.Chem. 69:2626-30 (1997), and Turgeon, “The Lab of the Future on CD-ROM?”Medical Laboratory Management Report. December 1997, p.1. Automatedhybridizations on chips or in a microfluidic environment arecontemplated methods of practicing the invention.

Although microfluidic environments are one embodiment of the invention,they are not the only defined spaces suitable for performinghybridizations in a fluid environment. Other such spaces includestandard laboratory equipment, such as the wells of microtiter plates,Petri dishes, centrifuge tubes, or the like can be used.

Another method for identifying the presence of SNPs is theoligonucleotide ligation assay (“OLA”), which may conveniently becoupled with flow cytometric analysis for rapid, accurate analysis ofSNPs. See, e.g., Iannone, M. A., et al., Cytometry, 39(2):131-40 (2000);and Jinneman, K. C., et al., J. Food Prot. 62(6):682-5 (1999). PCR andOLA can be used in tandem with yet another technique, Sequence-CodedSeparation, or “SCS,” to provide specificity, sensitivity, and multiplexcapability. See, e.g., Brinson, E. C., et al., Genet Test 1(1):61-8(1997) (erratum in Genet Test 2(4): 385 (1998)).

SNPs are also detected in the art by reverse dot blot allele-specificoligonucleotide (ASO) hybridization. See, e.g., Winichagoon, et al.Prenat Diagn 19:428-35 (1999), and Labuda et al., Anal Biochem 275:84-92(1999). One method asserted to be faster than ASO hybridization fordetecting single base pair changes is the so-called amplification ofrefractory mutation system, or “ARMS.” See, e.g., Bradley et al., GenetTest 2:337-41 (1998).

Mass spectrometry (“MS”) can also be used to detect SNPs. For example,matrix-assisted laser desorption-ionization-time-of-flight (“MALDI-TOF”)MS has been shown to be adaptable to high-throughput applications fordetecting SNPs. See, e.g., Griffin, T., and Smith, L., Trends Biotechnol18(2):77-84 (2000). A cost effective procedure for identifying SNPsusing MS is taught by Sauer, S., et al., Nucl Acids Res 28(5):E13 (March2000).

In addition to these gene-based techniques, a variety of techniques areavailable which screen for functional changes, specifically, byscreening for inhibition of monooxygenases. E.g., Crespi, C. L., et al.,Med. Chem. Res. 8(7/8):457-471 (1998); Crespi, C. L., et al., AnalBiochem 248(1):188-90 (1997). The latter reference provides afluorescent method for determining the IC₅₀ for a test compound anddetailed optimizations of the procedure for nine cytochrome P450 enzymesare set forth by GENTEST Corp. (Woburn, Mass.) which can be foundon-line by entering “www.” followed by “gentest.com”. Modification ofthis procedure for the enzyme encoded by the EtaA gene, using ETA as thesubstrate, will be readily apparent to persons of skill in the art. Inthese assays, the enzyme encoded by the wild-type EtaA gene (the“control enzyme”) is tested to determine the IC₅₀ of ETA. The enzymeencoded by the EtaA gene of a MTb of interest (the “test enzyme”), suchas that obtained in a biological sample from a person to be screened, isthen tested by the same procedure. A difference in the IC₅₀ of the testenzyme compared to that of the control enzyme indicates a mutation inthe gene.

Mutations in the gene can also be detected by detecting mutated forms ofthe protein encoded by the gene. A mutation that results in a truncatedprotein or one with a conformation other than that of the normal enzymecan be expected to have epitopes which are not present on the normalenzyme. These mutated forms of the enzyme can be used to raiseantibodies. Methods of producing polyclonal and monoclonal antibodiesare known to those of skill in the art. See, e.g., Coligan (1991)Current Protocols in Immunology Wiley/Greene, NY; Harlow and Lane (1989)Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites etal. (eds.) Basic and Clinical Immunology (4th ed.) Lange MedicalPublications, Los Altos, Calif.; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; Birchand Lennox, Monoclonal Antibodies: Principles and Applications,Wiley-Liss, New York, N.Y. (1995). Antibodies so raised are generallytested by being absorbed against the normal enzyme (conveniently, theenzyme is immobilized on a column and the antibodies run over thecolumn) to remove those which cross react with the form of the enzymeexpressed by the normal EtaA gene.

In another set of embodiments, mutations in the EtaA gene can bedetected by culturing MTb of interest, such as those isolated from abiological sample from a person being screened for resistant MTb, in amedium containing ETA and detecting whether the culture medium does ordoes not contain a metabolic product indicating that the monooxygenaseencoded by the EtaA gene is functional. For example, the tests candetect the metabolic product (2-ethyl-pyridin-4-yl)-methanol, which theresults herein establish for the first time is the product of ETAmetabolism by susceptible MTb. The presence of this product in theculture medium of MTb cultured with ETA indicates that the organismbeing tested is susceptible to ETA treatment; the absence of thisproduct in the medium indicates that the organism is resistant.Conveniently, a culture of a reference ETA-susceptible MTb is grown atthe same time as a control so that the presence or absence of themetabolic product in the medium of the MTb of interest can be comparedto that present in the medium from the control organism. In preferredforms, radiolabeled ETA is used and the presence of the radiolabeledproduct is detected in the test and reference cultures over a period oftime is detected. For example, if ¹⁴C-labeled ETA is added to a cultureof M. tuberculosis, the subsequent presence of ¹⁴C-labeled(2-ethyl-pyridin-4-yl)-methanol indicates that the organisms aresusceptible to ETA.

The presence of the metabolic product can be determined by any of anumber of analytic means known in the art. The Example section describesthe use of several of these methods, thin-layer chromatography (TLC)high-pressure liquid chromatography(HPLC), and mass spectrometry, toidentify (2-ethyl-pyridin-4-yl)-methanol as the major metabolic productof EtaA-encoded monooxygenase activity. Other techniques can, however,also be used to identify this metabolic product, such as raisingantibodies to (2-ethyl-pyridin-4-yl)-methanol by the methods discussedabove and using the antibodies to quantitate the presence or absence of(2-ethyl-pyridin-4-yl)-methanol in culture media by ELISAs. In apreferred embodiment, the determination is made by subjecting a samplefrom the culture to TLC in which a sample known to be(2-ethyl-pyridin-4-yl)-methanol is run as a control. Where the ETA hasbeen radioactively labeled, detection of the metabolic product can be bysubjecting the TLC to autoradiography. Immunoassays can also employchemiluminescence or electroluminescence detection systems. Such systemsinclude luminol, isoluminol, acridinium phenyl esters and otheracridinium chemiluminophores such as acridinium(N-sulphonyl)carboxamides, and ruthenium salts for the detection ofconventional enzyme-labelled conjugates. These agents are typically usedin ELISAs or in conjunction with a chemiluminescent substrate.

Methods for Amplification of the EtaA Gene or Portions Thereof

Some of the detection methods discussed above employ amplification ofthe EtaA gene. The EtaA gene or desired portions thereof can beamplified by cloning or by other in vitro methods, such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (SSR). These and other amplificationmethodologies are well known to persons of skill.

Examples of these techniques and instructions sufficient to directpersons of skill through cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in EnzymologyVol. 152, Academic Press, Inc., San Diego, Calif. (1987) (hereinafter,“Berger”); Sambrook et al., Molecular Cloning—A Laboratory Manual (2nded.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press,NY (1989), (“Sambrook et al.”); Ausubel, supra; Cashion et al., U.S.Pat. No. 5,017,478; and Carr, European Patent No. 0 246 864.

Examples of techniques sufficient to direct persons of skill throughother in vitro amplification methods are found in Berger, Sambrook, andAusubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCRProtocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (“Innis”); Arnheim &Levinson (Oct. 1, 1990) C&EN 36-47; J. NIH Res., 3: 81-94 (1991); Kwohetal., Proc. Natl. Acad. Sci. USA 86: 1173 (1989); Guatelli et al.,Proc. Natl. Acad. Sci. USA 87, 1874 (1990); Lomell et al. J. Clin.Chem., 35: 1826 (1989); Landegren et al., Science, 241: 1077-1080(1988); Van Brunt, Biotechnology, 8: 291-294 (1990); Wu and Wallace,Gene, 4: 560 (1989); and Barringer et al., Gene, 89: 117 (1990).

In one preferred embodiment, the MTb EtaA gene can be isolated byroutine cloning methods. The cDNA sequence of the gene can be used toprovide probes that specifically hybridize to the EtaA gene in a genomicDNA sample (Southern blot), or to the EtaA mRNA, in a total RNA sample(e.g., in a Northern blot), or to cDNA reverse-transcribed from RNA (ina Southern blot)). Once the target EtaA nucleic acid is identified(e.g., in a Southern blot), it can be isolated according to standardmethods known to those of skill in the art (see, e.g., Sambrook et al.,supra; Berger, supra, or Ausubel, supra).

In another preferred embodiment, the MTb EtaA cDNA can be isolated byamplification methods such as polymerase chain reaction (PCR). Oneexample of amplifying the MTb EtaA gene, including the primers used, isset forth in the Examples. Persons of skill in the art will recognizethat other sets of primers could readily be designed from the sequenceof MTb which would likewise amplify the EtaA gene.

In a particularly preferred embodiment, the EtaA gene can be amplifiedusing the primers 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3) and5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4) (EtaA). Manyother primer sets can be selected using standard programs widelyavailable in the art. For example, the program “Primer3” is availableon-line by entering “www-” followed by“genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi.” This program wasused to select the primer pairs noted above, using the defaultconditions. The program was also used to select the following sequencingprimers, which can be used to amplify sections of the EtaA gene forsequencing:

5′ ATCATCCATCCGCAGCAC 3′; (SEQ ID NO:5) 5′ AAGCTGCAGGTTCAACC 3′; (SEQ IDNO:6) 5′ GCATCGTGACGTGCTTG 3′; (SEQ ID NO:7) 5′ AAGCTGCAGGTTCAACC 3′;(SEQ ID NO:8) 5′ TGAACTCAGGTCGCGAAC 3′; (SEQ ID NO:9) 5′AACATCGTCGTGATCGG 3′; (SEQ ID NO:10) 5′ ATTTGTTCCGTTATCCC 3′; (SEQ IDNO:11) 5′ AACCTAGCGTGTACATG 3′; (SEQ ID NO:12) 5′ TCTATTTCCCATCCAAG 3;(SEQ ID NO:13) and 5′ GCCATGTCGGCTTGATTG 3′. (SEQ ID NO:14)Labeling of Nucleic Acid Probes

Where the EtaA DNA or a subsequence thereof or an mRNA of such DNA is tobe used as a nucleic acid probe, it is often desirable to label thesequences with detectable labels. The labels may be incorporated by anyof a number of means well known to those of skill in the art. However,in a preferred embodiment, the label is simultaneously incorporatedduring the amplification step in the preparation of the sample nucleicacids. Thus, for example, polymerase chain reaction (PCR) with labeledprimers or labeled nucleotides will provide a labeled amplificationproduct. In another preferred embodiment, transcription amplificationusing a labeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP)incorporates a label into the transcribed nucleic acids.

Alternatively, a label may be added directly to an original nucleic acidsample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplificationproduct after the amplification is completed. Means of attaching labelsto nucleic acids are well known to those of skill in the art andinclude, for example nick translation or end-labeling (e.g. with alabeled DNA) by kinasing of the nucleic acid and subsequent attachment(ligation) of a nucleic acid linker joining the sample nucleic acid to alabel (e.g., a fluorophore).

Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include biotin for staining with labeledstreptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescentdyes (e.g., fluorescein, texas red, rhodamine, green fluorescentprotein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P),enzymes (e.g., horse radish peroxidase, alkaline phosphatase and otherscommonly used in an ELISA), and colorimetric labels such as colloidalgold or colored glass or plastic (e.g., polystyrene, polypropylene,latex, etc.) beads. Patents teaching the use of such labels include U.S.Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

Kits

The invention further provides kits for determining the ability of a M.tuberculosis bacterium to metabolize a thioamide, thereby permitting adetermination of whether the bacterium is susceptible or resistant tothioamide- or thiocarbonyl-based agents. The kits can take any of avariety of forms, such as:

-   -   a kit for performing TLC to detect the presence of        (2-ethyl-pyridin-4-yl)methanol will usually provide a sample of        (2-ethyl-pyridin-4-yl)methanol which can be run in parallel with        the experimental sample to provide a positive control;    -   a kit may provide radiolabeled ETA so that the presence or        absence of a product of EtaA metabolism can be detected. For        example, the kit may provide ¹⁴C-labeled ETA so that the        presence or absence of labeled (2-ethyl-pyridin-4-yl)methanol        can be detected;    -   a kit may provide primers for amplifying an EtaA gene or a        portion thereof containing a mutation that affects the ability        of the bacterium to oxidize a thioamide, such as        5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3) and        5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4), or        any of the other primer pairs set forth above. It should be        noted that, due to the low synonymous rate of mutation of M.        tuberculosis, it is believed that all naturally-occurring        mutations in the EtaA gene will reduce the ability of the        organism to oxidize a thioamide. The kit may also include        isolated EtaA genes containing one or more mutations from the        wild-type sequence (SEQ ID NO:1), or nucleic acid sequences        derived from such an EtaA gene, for use as a positive control        during PCR or other amplification procedures;    -   a kit may provide one or more antibodies which permit the use of        ELISAs or other immunoassays known in the art. Typically, the        antibodies will be raised against        (2-ethyl-pyridin-4-yl)methanol, to permit detection of whether        this metabolic product is produced by a particular culture, or        antibodies against the gene product of the wild-type EtaA gene,        or against a gene product expressed from a missense, nonsense,        or frameshift mutation of the EtaA gene.

EXAMPLES Example 1 Synthesis of 2-ethyl-[¹⁴C]thioisonicotinamide(1-[¹⁴C]ETA)

2-ethylpyridine was converted to its N-oxide salt in almost quantitativeyield using 35% hydrogen peroxide in acetic acid and the correspondingN-oxide was subjected to a nitrating mixture of sulfuric and nitricacids to form 2-ethyl-4-nitropyridine N-oxide in 60% yield (Kucherova,et al., Zhurnal Obshchei Khimii 29:915-9 (1959). Reduction using ironfilings, hydrochloric, and acetic acid (Gutekunst and Gray, J. Am. ChemSoc., 44:1741 (1922)) allowed us to isolate 2-ethyl-4-aminopyridine,which was converted to 2-ethyl-4-bromopyridine through the perbromideusing 50% aqueous hydrobromic acid and sodium nitrite (Kucherova et al.,supra). The resulting bromide was heated with copper cyanide inN-methylpyrrolidin-2-one to afford 2-ethyl-4-[¹⁴C]cyanopyridine (Lawrieet al., J Labelled Compounds Radiopharmaceutic 36:891-8 (1995)).[¹⁴C]-copper cyanide was obtained from [¹⁴C]-sodium cyanide (AmershamPharmacia Biotech, Inc., Piscataway, N.J. 08855) using copper (II)sulfate pentahydrate and sodium sulfite (Sunay et al., J LabelledCompounds Radiopharmaceutic 36:529-36 (1995); Meinert et al., J LabelledCompounds Radiopharmaceutic 14:893-6 (1978)). The nitrite was convertedto 1-[¹⁴C]-ETA by hydrogen sulfide treatment and the resulting thioamidewas purified to 98% final radiochemical purity using normal phase HPLCwith a preparative ADSORBOSPHERE silica column (5μ, 300×22 mm, AlltechAssociates, Deerfield, Ill.) and an isochratic eluent of 90% chloroform,10% methanol. Unlabeled ETA synthesized using the same procedureco-chromatographed with commercially available ETA (Sigma-AldrichChemical Company, Milwaukee, Wis.) and showed correct analytical data.

Example 2 Materials and Methods for Determining in vivo Metabolism of1-[¹⁴C]ETA

The indicated mycobacterial species were grown in culture to an OD650 of1.0-1.5 and then concentrated 10-fold in middlebrook 7H9 broth media(DIFCO laboratories, Detroit, Mich.). The culture suspensions weretreated with 0.01 μg ml⁻1 of [¹⁴C]-ETA (55 mCi/mmol) and sequentialculture aliquots were removed at the indicated times, filtered and flashfrozen. Samples of 2 μl were analyzed by TLC on Silica gel 60 plates (EMScience, Gibbstown, N.J. 08027) developed with 95:5 ethylacetate:ethanol. Prior to spotting radioactive samples on TLC plates asmall amount of unlabeled ETA was spotted to circumvent silica-catalyzedair oxidation of the low concentration radioactive ETA samples.

Metabolites were identified by comparison with well characterizedsynthetic standards prepared as follows: the sulfoxide was prepared byhydrogen peroxide oxidation of ETA as previously described (Walter andCurtis, Chem Ber 93:1511 (1960)). The acid was made by reflux hydrolysisof the thioamide with 30% NaOH (Aq); ¹H-NMR (CDCl₃:CD₄OD;1:1); δ 1.26 t,2.83 q, 7.63 d, 7.71 s, 8.52 d; ES-MS (MH+) 152.1 m/e. The amide (4) wasmade by treating the corresponding acid chloride with ammoniumhydroxide; ¹H-NMR (CDCl₃); δ 1.34 t, 2.91 q, 7.45 d, 7.55 s, 8.65 d;ES-MS (MH⁺) 151.2 m/e. (2-ethyl-pyridin-4-yl)-methanol (5) was made byRedAl reduction of the acid in THF; ¹H-NMR (CDCl³); δ 1.28 t, 2.84 q,7.11 d, 7.19 s, 8.48 d; ¹³C-NMR (CDCl₃); 14.12, 30.54, 63.92, 118.73,119.64, 149.25, 150.60, 163.89; ES-MS (MH⁺) 138.0 m/e.

Cells from sequential culture aliquots from the metabolic conversionassays (volumes given in figure legends) were collected by filtrationonto 0.22 micron GS filter disks (Millipore, Bedford, Mass.) undervacuum on a Hoeffer apparatus and were washed twice with 0.1 mM sodiumphosphate, pH 7.5, 100 mM NaCl (500 μl). The cell associatedradioactivity was measured in 4 ml of EcoscintA scintillation solution(National Diagnostics, Atlanta, Ga.). HPLC separation of the [¹⁴C]-ETAmetabolite mixture was achieved using a reverse-phase LUNA column (5μ,C18(2), 250×4.6 mm, Phenomenex, Torrence, Calif.) with a gradient of:(0-5 min) 0% acetonitrile, 100% water; then (5-65 min) to 70%acetonitrile; then (65-80 min) to 100% acetonitrile (all solventscontained 0.1% trifluoroacetic acid). The retention time of the unknownradiolabeled major metabolite (5) utilizing continuous radiodetection(β-RAM, INUS Systems, Florida), was used to guide cold large scale ETAfeeding experiments with up to 1 liter of log phase MTb H37Rv, to whichwe fed 10 μg ml⁻ 1 ETA (Sigma-Aldrich, Milwaukee, Wis.). We HPLCisolated very small quantities of unlabeled metabolite with a similarretention time to (5), utilizing UV₂₅₄ detection. The metabolite (5)gave a mass of 137 (137.9 MH+)(Mass spectrometer model API300TQMS,Perkin Elmer/Sciex, Toronto, Canada). For Mycobacterium smegmatis (“MSm”or “MSMEG”), macromolecule associated radioactivity was determined byresuspending cells from micro-centrifuged 900 min aliquots (400 μl) inPBS. The cells were ruptured by bead-beating (MiniBeadBeater, BioSpecProducts, Bartlesville, Okla., 3×45 sec, 0.1 mm glass beads) andextensively dialyzing the lysates with centricon 10 concentrators(Amicon Inc, Beverly, Mass.) before analysis in 4 ml of EcoscintAscintillation solution.

Example 3 Cloning of EtaA and EtaR

Genomic DNA from MTb H37Rv was partially digested with Sau3AI (NewEngland BioLabs, Beverly, Mass.) to give fragments of various sizes.Fragments ranging from 1 Kb to 10 Kb were ligated to pMV206Hyg (Mdluliet al., J Infect Dis 174:1085-90 (1996)) that had been previouslylinearized with BamHI (New England BioLabs). The ligation mixtures wereelectroporated into Escherichia coli DH5α (Life Technologies, GrandIsland, N.Y.) for amplification of the DNA library which wassubsequently purified and electroporated into MTb H37Rv. The resultingtransformants were plated on 7H11 (DIFCO) agar plates that containedHygromycin (Life Technologies, 200 μg ml⁻¹) and the indicatedconcentrations of ETA. Five colonies were isolated that had MICs for ETAfrom 2.5 to 5.0 μg/ml (the MIC for wild type MTb is 1.0 μg/ml) (Rist,Adv Tuberc Rec 10:69-126 (1960)).

EtaA and EtaR were PCR-amplified from H37Rv chromosomal DNA using thefollowing primers 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3) and5′-ATAAGAATGCGGCCGCAACCGTGCTAAAGCTAAACC-3′ (SEQ ID NO:4) (Rv3854c,EtaA); 5′-GGGGTACCGCACACTATCGACAC GTAGTAAGC-3′ (SEQ ID NO:15) and5′-ATAAGAATGCGGCCGCGCGGTTCTC GCCGTAAATGCT-3′ (SEQ ID NO:16) (EtaR) andinserted directionally into KpnI and NotI digested pMH29 (Mdluli et al.,supra).

Example 4 Sequence Analysis of ETA-Resistant Clinical Isolates

Using the primers described in the previous Example, EtaA was PCRamplified from genomic DNA containing, drug resistant isolate lysates (1ml, bead beaten for 3×45 sec and aqueous diluted 10-fold). EtaA wassequenced in entirety by primer walking for all isolates (SEQWRIGHT Inc,Houston, Tex.) and observed mutations were confirmed on both strands.For the three isolates without mutations in EtaA, EtaR and theintergenic region were also sequenced in entirety without observing anymutations.

Example 5 Synthesis and in vivo Metabolism of [14C]-ethionamide

We synthesized [¹⁴C]-ETA from 2-ethylpyridine and [¹⁴C]-sodium cyanide(see Example 1, supra) to study the metabolism of ETA by whole cells ofMTb. In the presence of live cells of MTb, ETA is converted through theS-oxide (2) to a single major metabolite (5) as seen by TLC analysis ofsequential time points (FIG. 1A). Metabolites corresponding to theS-oxide (2), nitrile (3), and the amide (4) were identified bycochromatography (TLC and HPLC) with standards synthesized by knownmethods and characterized by 1H-NMR, 13C-NMR and mass spectrometry.These metabolites were produced in small amounts by the cellularoxidation of ETA but they were the dominant products of air oxidation ofETA (compare lanes h and i in FIG. 1A).

In contrast, metabolite 5 was only produced by live cells of MTb and wasnot seen upon air oxidation of ETA. The thioamide S-oxide 2 wastransiently produced in whole cells and appeared temporally to be aprecursor of metabolite 5 (FIG. 1B). Cold ETA feeding experimentsallowed the isolation of unlabeled metabolite 5 which displayed amolecular mass of 137 by LC-MS (FIG. 1C). We assigned this metabolite as(2-ethyl-pyridin-4-yl)-methanol (5) and confirmed this byco-chromatography (TLC and HPLC) with an authentic synthetic alcoholstandard. The upper HPLC trace in FIG. 1C shows the continuousradio-detector output from a sample corresponding to [1-¹⁴C]ETA that hasbeen air oxidized in media (lane i in FIG. 1A). The lower trace shows asample from MTb metabolism of [1-¹⁴C]ETA after 1.5 hr of exposure (laned in A). The UV₂₅₄ trace of synthetic (2-ethyl-pyridin-4-yl)methanol issuperimposed in gray.

The production of metabolite (5) from ETA by tuberculosis is surprisingas 4-pyridylmethanol is a major metabolite of INH by whole cells of MTb(Youatt, J. Aust J Chem 14:308 (1961); Youatt, J. Aust J Exp Biol MedSci 38:245 (1960); Youatt, J. Aust J Biol Med Sci 40:191(1962)). Likespontaneous oxidation of INH, spontaneous oxidation of ETA fails toproduce any trace of the major in vivo metabolite,(2-ethyl-pyridin-4-yl)methanol. INH has been shown to be activated byKatG in vitro to a variety of products including isonicotinic acid,isonicotinamide and isonicotinaldehyde (which in vivo is rapidly reducedto 4-pyridylmethanol) (Johnsson, K. et al., J Am Chem Soc 116:7425(1994)). INH metabolism to 4-pyridylmethanol only occurs indrug-susceptible organisms while drug-resistant organisms no longerproduce this metabolite (Youatt, J., Am Rev Respir Dis 99:729 (1969)).Similarly, we postulate that ETA is activated via the correspondingS-oxide to a sulfinate that can form an analogous aldehyde equivalent(an imine) through a radical intermediate (Paez, O. A. et al., J OrgChem 53:2166 (1988)).

Example 6 Identification of a Monooxygenase that Activates ETA

To elucidate the enzymatic basis for activation of ETA to metabolite 5by MTb we selected for ETA resistance in MTb by transformation of a 1-10kb insert-containing library of MTb chromosomal DNA in pMV206Hyg (Georgeet al., J. Biol. Chem. 270:27292-8 (1995)). Five colonies were isolatedthat had MICs for ETA from 2.5 to 5.0 μg/ml (the MIC for wild type MTbis 1.0 μg/ml). Upon restriction analysis the five independent plasmidswere shown to contain the same genomic region on different overlappingSau3AI fragments. This cloning was also done with genomic DNA from astrain reported to be ETA-resistant but the same genomic locus wasobtained with no alterations compared to H37Rv, suggesting that theresistance was not associated with alterations to this region but simplywith its overexpression. The common region to all theresistance-conferring clones encompassed only one gene (Rv3855, EtaR)that showed broad homology to many TetR family transcriptionalregulators. A 76 nt intergenic region separates this putative regulatorfrom a divergently transcribed monooxygenase (Rv3854c, EtaA). One of theisolated library plasmids containing only the etaR gene waselectroporated into MTb and MSm and the resulting MTb transformants grewas a lawn at 2.5 and 5 μg/ml ETA indicating that EtaR was solelyresponsible for ETA resistance. The MSm transformants were able to growat greater than 200 μg/ml ETA, compared to growth of vector controlcontaining MSm at 50 μg/ml.

Two other monooxygenase/regulator pairs with similar genomicorganization appeared to have high homology in both the regulator andmonooxygenase components to the MTb locus, one from Dienococcusradiodurans (White et al., Science 286:1571-7 (1999)) and the other fromStreptomyces coelicolor (Redenbach et al., Mol Microbiol 21:77-96(1996). This conservation suggested that the effect of regulatorexpression was to modulate production of the adjacent monooxygenase. Tosee if EtaR-mediated repression of EtaA was the cause of ETA resistance,we transformed MTb and MSm with pMH29 plasmid constructs containing etaRand EtaA separately under the control of a strong constituitive promoter(Mdluli et al., supra). Although we could observe resistance with EtaRconstructs in MTb, we were not successful in overexpressing EtaA in MTb,suggesting expression of this enzyme is tightly controlled in thisorganism. MSm overexpressing the putative repressor was found to be ETAresistant with a measured MIC greater than 62.5 μg/ml on solid media(FIG. 2A). Although the recombinant MSm were equally susceptible tokilling with INH, the bacteria overexpressing EtaA were found to behypersensitive to ETA with noticeable growth inhibition at 2.5 μg/ml, alevel comparable to the normal MIC for MTb (FIG. 2A). Qualitativelycomparable results were obtained when these organisms were treated withETA S-oxide (although the absolute MIC for the sulfoxide is lower, EtaRconferred resistance and EtaA conferred hypersensitivity). These resultssuggest that EtaA is directly responsible for thioamide S-oxideoxidative activation and that EtaR modulates expression of this enzyme.

Example 7 Effect of the EtaR Gene

To link expression of the EtaA activator more directly with ETAmetabolism we examined [¹⁴C]-ETA conversion by whole cells of the MSmtransformants described above over a time-course study as shown in FIG.3. The EtaA overproducing MSm was found to convert ETA to metabolite 5much more quickly than vector control (FIG. 3A). Although the EtaRoverproducing strain did appear to effect this conversion lessefficiently than the control, the result was not dramatic since MSmnormally only weakly activates ETA consistent with this organism'shigher overall MIC for ETA (FIG. 1B). These studies directly correlateETA activation and metabolism with toxicity as measured by MIC. Tounderstand the effect of drug activation we also examined covalentincorporation of [¹⁴C]-ETA into cellular macromolecules by lysingtreated cells and then extensively dialyzing away small molecules. Drugactivation was found to correlate directly with incorporation of labeleddrug into macromolecules (FIG. 2D).

Example 8 Correlation of ETA-Resistance with Resistance to otherThioamide Drugs

ETA is only one example of a thiocarbonyl-containing antituberculosismedication approved for clinical use. Among the second-line tuberculosistherapeutics there are two other such molecules, thiacetazone (11) andthiocarlide (isoxyl) (12) (FIG. 4A) that might be similarly activated byEtaA-catalyzed S-oxidation. To elucidate the clinical relevance ofEtaA-mediated resistance to thiocarbonyl-containing drugs as a class wecharacterized a set of 14 multidrug resistant isolates from patients inCape Town, South Africa. These isolates were selected on the basis ofthiacetazone resistance and then characterized with respect to ETAresistance. Eleven of fourteen of these isolates were found to be ETAcross-resistant. Despite the fact that none of the patients had beentreated with thiocarlide, thirteen out of fourteen of the isolatesshowed thiocarlide cross-resistance.

To examine at the molecular level the relevance of EtaA-mediatedthiocarbonyl activation for this class of compounds, we PCR-amplifiedand sequenced the EtaA gene from all 14 multidrug-resistant patientisolates. In addition, we examined an in vitro generated ETAmono-resistant strain (ATCC 35830). Eleven of 14 clinical isolates hadamino acid altering mutations in EtaA, as indicated in FIG. 4B.

EtaA was PCR amplified from chromosomal DNA-containing lysates of 1 mlcultures of patient isolates using the primers set forth in Example 1,above. EtaA was sequenced in its entirety by primer walking for allisolates and observed mutations were confirmed on both strands. For thethree isolates without mutation in EtaA, EtaR and the intergenic regionwere also sequenced in their entirety without observing any mutations.Eleven of fourteen clinical isolates had amino acid-altering mutationsin EtaA, as indicated in FIG. 3A. The nucleotide change at base 1025 wasfound in two isolates, that at base 1141 in three isolates. Along withthe single nucleotide changes, a 1 nt nucleotide deletion (at base 65)and addition (at base 811) were found. In the ATCC ETA mono-resistantstrain, a nucleotide change at position 557 of EtaA was found. Thepatient isolates in which mutations could not be found (either in EtaA,EtaR or their promoter regions) were subsequently tested and found to befully sensitive to ETA. Thus there is a 100% correspondence betweenmutation in EtaA and ETA cross-resistance among thesethiacetazone-resistant strains.

Example 9 Mechanism of ETA Activation

INH (6) has been shown to be activated by KatG in vitro to a variety ofproducts including isonicotinic acid, isonicotinamide andisonicotinaldehyde (9) (which in vivo is rapidly reduced to4-pyridylmethanol (10)) (Johnsson, K. & Schultz, P. G., J Am Chem Soc116:7425-68 (1994)). The results support the notion that in vivo INH ismetabolized by oxidation to an acyl diimide (7), then to a diazonium ion(8) or an isonicotinyl radical which may abstract a hydrogen atom from asuitable donor to form isonicotinaldehyde. Similarly, we postulate thatETA is activated via the corresponding S-oxide (2) to a sulfinate thatcan form an analogous aldehyde equivalent (an imine) through a radicalintermediate. Hydrolysis of this imine could be followed by reduction ofthe resulting aldehyde to the observed metabolite (5).

The mechanistic linkage of the activated form of ETA and INH explains,in part, the observation that they share a final common target. Thestriking observation that both drugs give rise to essentially the samefinal metabolite upon productive activation of the drug, furthersubstantiates this common mechanism. Despite this commonality, an acylhydrazide and a thioamide must undergo very different activationprocesses by discrete enzymes before they converge upon an analogousreactive intermediate. The association of KatG with INH activation hasbeen firmly established by a combination of loss of activity studies,laboratory-selected drug-resistant mutants, overexpression, andclinically relevant mutations. The results here establish that EtaA isthe analogous enzyme for the activation of ETA and provide similarevidence based upon genetic manipulation of the enzyme levels andmutations observed in patient isolates.

Example 10 Relationship of EtaA to other Bacterial Enzymes

EtaA has two closely related homologs (Rv3083, Rv0565c) encoded withinthe MTb genome that share almost 50% identity to this monooxygenase(Cole, et al., Nature 393:537-44 (1998)). It is also a member of afamily of 14 more loosely related proteins, the majority of which areprobable monoxygenases. In addition, MTb has twenty additional homologsof Cytochrome P-450 containing oxygenases, the largest number everidentified within a single bacterial genome (Nelson, D. R., Arch BiochemBiophys 369:1-10 (1999)). The reason for this amazing radiation ofoxidative enzymes is not clear but they may improve bacterial survivalin the face of various xenobiotic substances. In this vein, the ETAsusceptibility of this organism may arise from accidental activation byan enzyme intended to help detoxification.

Thiacetazone (11) has been widely used as a front-line therapeutic inAfrica and throughout the developing world because it is extremelyinexpensive. Although thiocarlide (12) has not been widely used there isrenewed interest in this drug and new analogs. There is an impressiveclinical history of cross-resistance among this set of three second-linetherapies. This cross-resistance suggested a common mechanism ofactivation of thiocarbonyl containing molecules that might allow thesimultaneous acquisition of drug resistance to this class oftherapeutic. When we examined patient isolates from Cape Town forcross-resistance to other thioamides or thioureas, we noted that thevast majority of ETA/thiacetazone resistant isolates were alreadyresistant to thiocarlide, despite the fact that these patients werenever treated with this drug.

The extensive cross-resistance among these compounds predicts multipleoverlapping mechanisms of resistance among clinically usedantituberculars: target associated between INH and ETA, andactivation-associated between ETA, thiacetazone, and thiocarlide. Suchconsiderations complicate appropriate drug therapy for the treatment ofmultidrug-resistant tuberculosis and these results provide an importanttool to help understand and quickly characterize the resistancemechanisms operating in a single patient, which may prove vital to apositive outcome.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of determining the ability of a Mycobacterium tuberculosisbacterium to oxidize a thioamide or a thiocarbonyl, said methodcomprising detecting a mutation in an EtaA gene (SEQ ID NO:1) in saidbacterium by detecting a product of said gene altered from the sequenceof SEQ ID NO:2, wherein detection of the gene product alteration isindicative of decreased ability to oxidize a thioamide or athiocarbonyl.
 2. The method of claim 1, wherein the gene productalteration results from a frameshift mutation selected from the groupconsisting of: a deletion at position 65, an addition at position 557,and an addition at position
 811. 3. The method of claim 1, wherein thegene product alteration results from a single nucleotide polymorphism.4. The method of claim 3, wherein the single nucleotide polymorphismcauses an amino acid substitution selected from the group consisting of:G43C, P51L, D58A, Y84D, T186K, T342K, and A381P.
 5. A method of claim 1,wherein said mutation is detected by specifically binding an antibody toa mutated product of the EtaA gene, wherein the specific binding of theantibody to the mutated gene product is indicative of a mutation whichinhibits the ability of the bacterium to oxidize a thioamide.
 6. Amethod of claim 1, wherein said gene product is in, or is isolated from,sputum.
 7. A method of claim 5, wherein detection of said specificbinding of said antibody and said mutated gene product is by ELISA.
 8. Amethod of claim 1, wherein said thioamide or thiocarbonyl is selectedfrom the group consisting of etbionamide, thiacetazone, and thiocarlide.9. A method of claim 1, wherein said mutation is detected by (a)culturing said bacterium in the presence of ethionamide; and (b) testingfor the presence or absence of (2-ethyl-pyridin-4-yl)methanol, whereinthe absence of (2-ethyl-pyridin-4-yl)methanol indicates that thebacterium has a mutation which is indicative of decreased ability tooxidize a thioamide.
 10. A method of claim 9, wherein the presence orabsence of (2-ethyl-pyridin-4-yl)methanol is tested by subjecting amedium in which the bacterium is cultured, or the bacterium, to analysisby thin-layer chromatography, high pressure liquid chromatography, ormass spectrometry.
 11. A method of claim 9, wherein the ethionamide ofstep (a) is radioactively labeled.
 12. A method of claim 9, wherein the(2-ethyl-pyridin-4-yl)methanol is radioactively labeled.
 13. A method ofclaim 1, wherein said mutation is detected by specifically binding anantibody to a mutated product of the EtaA gene selected from the groupof mutations consisting of (a) a frameshift mutation consisting of adeletion at position 65, an addition at position 557, and an addition atposition 811, and (b) a single nucleotide polymorphism which causes anamino acid substitution selected from the group consisting of: G43C,P51L, D58A, Y84D, T186K, T342K, and A381P, wherein the specific bindingof the antibody to the mutated gene product is indicative of a mutationwhich inhibits the ability of the bacterium to oxidize a thioamide.